Indexed library of cells containing genomic modifications and methods of making and utilizing the same

Abstract

Methods and vectors (both DNA and retroviral) are provided for the construction of a Library of mutated cells. The Library will preferably contain mutations in essentially all genes present in the genome of the cells. The nature of the Library and the vectors allow for methods of screening for mutations in specific genes, and for gathering nucleotide sequence data from each mutated gene to provide a database of tagged gene sequences. Such a database provides a means to access the individual mutant cell clones contained in the Library. The invention includes the described Library, methods of making the same, and vectors used to construct the Library. Methods are also provided for accessing individual parts of the Library either by sequence or by pooling and screening. The invention also provides for the generation of non-human transgenic animals which are mutant for specific genes as isolated and generated from the cells of the Library.

1 - 28 . (canceled) 
     
     
         29 . A method of identifying an ES cell clone having a mutation in a selected gene from a collection of ES cell clones having nonspecific mutations in the genome, comprising:
 a) providing at least two pools of ES cell clones having nonspecific mutations in the genome, wherein each pool comprises at least two samples wherein each sample comprises at least 1 ES cell clone, and wherein each pool comprises at least 16 ES cell clones;   b) screening the at least two pools of ES cell clones to identify one or more pools comprising an ES cell clone having a mutation in the selected gene; and   c) identifying an ES cell clone having a mutation in the selected gene.   
     
     
         30 . The method of  claim 29 , wherein the screening comprises a method selected from PCR and hybridization. 
     
     
         31 . The method of  claim 29 , wherein identifying one or more pools comprising an ES cell clone having a mutation in the selected gene results in identifying an ES cell clone having a mutation in the selected gene. 
     
     
         32 . The method of  claim 29 , wherein each sample is at least one well of a multiwell plate. 
     
     
         33 . The method of  claim 32 , wherein identifying an ES cell clone having a mutation in the selected gene comprises identifying a well of a multiwell plate based on which pools of ES cell clones comprise an ES cell clone having a mutation in the selected gene. 
     
     
         34 . The method of  claim 29 , wherein identifying an ES cell clone having a mutation in the selected gene comprises separating at least one sample comprising a plurality of ES cell clones having mutations in the genome into individual ES cell clones and screening the individual ES cell clones to identify the ES cell clone having a mutation in the selected gene. 
     
     
         35 . The method of  claim 34 , wherein the screening the individual ES cell clones comprises a method selected from PCR and hybridization. 
     
     
         36 . The method of  claim 29 , wherein identifying an ES cell clone having a mutation in the selected gene comprises:
 a) identifying at least one sample comprising a plurality of ES cell clones having mutations in the genome, wherein the plurality includes an ES cell clone having a mutation in the selected gene, based on which pools of ES cell clones comprise an ES cell clone having a mutation in the selected gene;   b) separating the plurality of ES cell clones of the sample into individual ES cell clones; and   c) screening the individual ES cell clones to identify the ES cell clone having a mutation in the selected gene.   
     
     
         37 . The method of  claim 36 , wherein the at least one sample is at least one well of a multi-well plate. 
     
     
         38 . The method of  claim 36 , wherein the at least one sample comprises at least 50 ES cell clones having mutations in the genome. 
     
     
         39 . The method of  claim 36 , wherein the screening comprises a method selected from PCR and hybridization. 
     
     
         40 . The method of  claim 29 , wherein identifying an ES cell clone having a mutation in the selected gene comprises:
 a) identifying at least one sample comprising a plurality of ES cell clones having mutations in the genome, wherein the plurality includes an ES cell clone having a mutation in the selected gene, based on which pools of ES cell clones comprise an ES cell clone having a mutation in the selected gene;   b) separating the plurality of ES cell clones of the sample into individual ES cell clones;   c) forming at least two pools of individual ES cell clones;   d) screening the at least two pools of individual ES cell clones to identify one or more pools of individual ES cell clones that comprise an ES cell clone having a mutation in the selected gene; and   e) identifying an ES cell clone having a mutation in the selected gene based on which pools of individual ES cell clones comprise an ES cell clone having a mutation in the selected gene.   
     
     
         41 . The method of  claim 40 , wherein the at least one sample is at least one well of a multi-well plate. 
     
     
         42 . The method of  claim 40 , wherein the sample comprises at least 50 ES cell clones having mutations in the genome. 
     
     
         43 . The method of  claim 40 , wherein the screening comprises a method selected from PCR and hybridization. 
     
     
         44 . The method of  claim 29 , wherein each pool of ES cell clones in (a) comprises at least 24 ES cell clones. 
     
     
         45 . The method of  claim 29 , wherein each pool of ES cell clones in (a) comprises at least 50 ES cell clones. 
     
     
         46 . The method of  claim 29 , wherein each pool of ES cell clones in (a) comprises at least 1200 ES cell clones. 
     
     
         47 . The method of  claim 29 , wherein at least one pool of ES cell clones in (a) is made by pooling the wells of at least one column of at least one 96-well plate. 
     
     
         48 . The method of  claim 29 , wherein at least one pool of ES cell clones in (a) is made by pooling the wells of at least one row of at least one 96-well plate. 
     
     
         49 . The method of  claim 29 , wherein at least one pool of ES cell clones in (a) is made by pooling all of the wells of at least one 96-well plate. 
     
     
         50 . The method of  claim 29 , wherein each ES cell clone having a nonspecific mutation in the genome comprises a vector inserted nonspecifically into its genome. 
     
     
         51 . The method of  claim 50 , wherein the vector is a viral vector. 
     
     
         52 . The method of  claim 51 , wherein the vector is a retroviral vector. 
     
     
         53 . The method of  claim 50 , wherein the vector is a gene trapping vector.